Antimicrobial Resistance

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Antimicrobial Resistance

• 9.21.12

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Site of Action of antibiotics

• Inhibition of nucleic acid synthesis (Rifampin
  quinilones)
• Inhibition of protein synthesis (Tetracyclines
  Chloramphenicol, macrolides, clindamycin,
  aminoglycosides, linezolid)
• Action on cell membrane (Polyenes Polymyxin)
• Interference with enzyme system (Trimethoprim,
  Sulphamethoxazole)
• Action on cell wall (Penicillin cephalosporins,
  Vancomycin, carbapenams)

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Mechanisms of Drug Resistance

• Change in drug target
• Production of an enzyme that modifies or
  inactivates the agent
• Reduced accumulation of the agent
• Limited uptake
• Active Efflux
• Loss of a pathway involved in drug activation

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Mechanisms of Drug Resistance
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Mechanisms of Drug Resistance
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Mechanisms of Gram-Negative Bacterial Resistance
to Antibiotics
Antibiotic Class Mechanism of Resistance
Cephalosporins ESBLs chromosomal cephalosporinases
?-Lactamase inhibitors hyperproducers of ?-lactamases new ?-lactamases resistant to inhibitors chromosomal cephalosporinases
Carbapenems porin mutations efflux pump overproduction (excluding imipenem) zinc metalloenzymes and other ?-lactamases
Fluoroquinolones alterations in DNA topoisomerase efflux mechanisms permeability changes
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Selection for antimicrobial-resistant Strains
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Target Alterations

• PBPs in cell membrane
• S. pneumoniae, MRSA
• Intrinsic resistance, enterococci, gonococci, H.
  infl
• D-Ala-D-Ala target VRE
• VanA, VanB, VanC, VanD
• Alterations in ribosomes
• Cell membrane changes

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Protein Binding Proteins

• Target for all B-lactams
• found as both membrane-bound and cytoplasmic
  proteins
• all involved in the final stages of the synthesis
  of peptidoglycan, which is the major component of
  bacterial cell walls
• More common R mechanism for gram positive
  organisms
• Gram neg access to PBP is limited by outer
  membrane and thus other mechanisms supersede the
  binding to this target

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Enzyme Production

• Aminoglycoside modifying enzymes
• B-lactamases
• Four structural classes
• Class A R of S aureus to penicillin, R of E coli
  to ampicillin and cephalothin plasmid mediated
• Class B hydrolyze carbapenmens/pens/cephs
  -chromosomal
• Class C chromosomal, active against
  cephalosporins
• Class D plamid mediatated
• ESBL K. pneumoniae, E. coli Derived from
  transfer of chromosomal genes for inducible amp C
  onto plasmids

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B-lactamase
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B-lactame ring
Cefipime
Increased stability to B-lactamase
Increased penetration into gram-positive
Ceftriaxone
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?-Lactamases Overview

• Large, diverse family of enzymes
• Widely dispersed in gram-positive (chromosoaml
  and plasmid) and gram-negative pathogens
  (plasmid)
• Major mechanism of resistance to ?-lactams in
  gram-negative pathogens
• Wide range of activity older enzymes hydrolyze
  older drugs, new derivatives have evolved for new
  drugs
• ESBLs
• AmpC ?-lactamases
• carbapenemases

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?-Lactamases

• Major groups for gram-neg
• TEM-wide spread-plasmid and transposon
• Enterobacteriaceae, Pseudomonas aeruginosa,
  Haemophilus influenzae, and Neisseria gonorrhoeae
  
• SHV-1
• Klebsiella pneumoniae (chromosomal) and E. coli
  (plasmid)
• Confer resistance to penicillins and first/second
  generation cephalosporins

b-lactamase
Extended spectrum-b-lactamase
TEM, SHV
1960 TEM-1
CTX
SHV
1980s Cefotaxime
TEM-2
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ESBL-Mediated Resistance

• Contain a number of mutations that allow them to
  hydrolyze expanded-spectrum ß-lactam antibiotics
• Derived from older antibiotic-hydrolyzing
  ?-lactamase enzymes (TEM-1, TEM-2, SHV-1)
• a single amino acid substitution can give rise to
  new ESBLs
• Not as catalytically efficient
• Inhibited by ß-lactamase inhibitors
• Susceptible to cefoxitin and cefotetan in vitro
  only
• 1040 of K pneumoniae, E coli express ESBLs

Rupp ME et al. Drugs. 200363353365.
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CTM-X predominant mechanism
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E. Coli predominant organism
Canton, Cur Opin in Micr 2006, Pages 466475
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Coresistances among the Enterobacteriaceae
isolates of the different ESBL types.
Morosini M et al. Antimicrob. Agents Chemother.
2006502695-2699
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Amp-C

• Confer resistance cephamycins (cefotetan,
  cefoxitin) and oxyimino- -lactams (cefotaxime,
  ceftriaxone, ceftazidime)
• Chromosomal in SPACE organisms and are inducible
• Poorly expressed in E. coli and is missing from
  klebsiella and salmonella species
• Plasmid mediated on other gram-neg, usually not
  inducible
• Not susceptible to inhibitors

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AmpC- vs ESBL-Mediated Resistance

• Different phenotypic characteristics
• AmpC type ?-lactamases typically encoded on
  chromosome of gram-negative bacteria, can also be
  found on plasmids
• AmpC type ?-lactamases hydrolyze broad- and
  extended-spectrum cephalosporins
• ESBLsNOT AmpC ?-lactamasesare inhibited by
  ?-lactamase inhibitors (eg, clavulanic acid)
• AmpC production is less effective on cefipime so
  best cephalosporin to test

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New CLSI Laboratory Standards

• Previously testing for ESBL was based on high MIC
  to oxyimino-beta-lactam substrates (cetriaxone,
  cefotaxime, cefipime, cetaz) and susceptibility
  to inhibitors followed by a confirmatory test to
  detect the enzyme
• Low sensitivity when mixed mechanisms at play, ie
  false positive results, some attempts to overcome
  this with cloxacillin-containing MullerHinton
  agar, which inhibits AmpC activity
• When ESBL present susceptibility changed to
  resist for penicillins, cephalosporins and
  monobactams
• Current practice MICs were changed
• 1-3 doubling dilutions lower
• No need for confirmation of enzyme
• No change in reporting

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Epidemiology of Plasmid AmpC Enzymes in the
United States

• Alvarez et al examined a sample of 752 resistant
  K pneumoniae, K oxytoca, and E coli strains
  from 70 sites in 25 US states
• Plasmids encoding AmpC-type ?-lactamase were
  found in
• 8.5 K pneumoniae samples
• 6.9 K oxytoca samples
• 4 E coli samples

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Carbapenemases

• beta-lactamases with versatile hydrolytic
  capacities.
• Ability to hydrolyze penicillins, cephalosporins,
  monobactams, and carbapenems.
• 2 major groups
• Metallo-b-lactamases (MBLs)
• Major R in pseudomonas, acinetobacter, and
  enterobacter
• Confer High level of R
• Serine b-lactamases
• Oxacillinases or D b-lactamases (OxaA)
• Not as Diverse
• Found mostly in acinetobacter
• Confer only low level of hydrolytic activity
  therfore another R is necessary to raise MIC
• Class A carbapenemases
• Found in pseudomonas and enterobacter, but
  predominant type is found on a plasmid in
  Klebsiella

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Mechanisms of Bacterial Resistance to
Fluoroquinolones

• Mutations in DNA gyrase and topoisomerase
• Overexpression of efflux pump system
• Bacterial membrane permeability changes

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Mechanisms of Antibiotic Resistance in
Nonfermenters

• P aeruginosa and Acinetobacter often multidrug
  resistant1
• Mechanisms of resistance include1,2
• production of ESBLs or AmpC b-lactamases
• increased efflux of antibiotic agent
• decreased outer membrane permeability
• DNA gyrase mutations
• aminoglycoside modifying enzymes

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Carbapenems Resistance Issues

• Mechanisms of resistance to carbapenems in P
  aeruginosa involve
• loss of OprD protein (initially called D2 porin)
• overproduction of efflux pump system
  (MexA-MexB-OprM)
• upregulation of other efflux system may be
  involved (cross-resistance to fluoroquinolones)
• Resistance to meropenem depends on both
• Resistance to imipenem mainly mediated through
  loss of OprD

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Carbapenems Resistance Issues
Carbapenem nucleus
Ertapenem
Imipenem
Mutated or missingD2 porin
D2 Porin (OprD)
Outer membrane
Periplasm
Penicillin-binding proteins (PBPs)
Cytoplasmic membrane
PBP1
PBP2
PBP3
PBP4
PBP5
Courtesy of John Quinn, MD.
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Mechanisms of Carbapenem Resistance
Impermeability

• OprD forms narrow transmembrane channels that are
  normally accessible only to carbapenems, not to
  other ß-lactams
• Loss of OprD porin is associated with decreased
  permeability of carbapenems and increased
  carbapenem MICs, whereas other ß-lactams remain
  active

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Mechanisms of Carbapenem Resistance Efflux
Systems in P aeruginosa

• Upregulation of MexAB-OprM efflux system
• associated with increased MICs of meropenem, not
  imipenem
• Coregulation of MexE-MexF-OprN efflux system with
  OprD porin in P aeruginosa
• upregulation of efflux associated with OprD
• associated with increased MICs of
  fluoroquinolones as well as carbapenems
• mechanism sometimes selected by fluoroquinolones,
  rarely by carbapenems

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MRSA

• Methicillin resistance is acquired via Mec A
• mobile chromosomal element called staphylococcal
  cassette chromosome (SCCmec)
• SCCmec types I, II, and III and are multidrug
  resistant-large cassettes
• Health-care associated
• SCCmec type IV and type V not multidrug resistant
• Community associated

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MecA

• Encodes penicillin binding protein (PBP) 2a
• Weak affinity for methicillin and all
  beta-lactams
• Substitutes for the usual PBP 1-3 that have a
  high affinity for beta-lactams
• Speculation of origination from CoNS

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S. Pneumoniae

• Pencillin
• Decreased affinity to PBP
• Can be overcome with high dose
• Macrolides
• Genetic changes to binding target on
  ribosome-high level can not be overcome erm(B)
• Efflux pump-lower level-may be overcome mef (A)
• Clindamycin
• Ribosomal methylation changing target erm(B)

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S. pneumoniae

• Fluoroquinilones
• Bind to either gyrase or topoisomerase or both
• Resistance from mutations in gyrA or parC
• reduce binding of the drug to the site of
  activity
• Mutations are step wise
• One mutation and R to cipro and levo
• More than one needed for gemi and moxi
• Tetracyclines
• Proteins are produced that package the drug into
  vessicles which are extruded from the cell

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Enterococcus

• Intrinsic (chromosomal, naturally occurring)
  resistance to
• B-lactam
• 10 to 1000 times more drug to inhibit an average
  Enterococcus than an average Streptococcus
• Due to penicillinase production and PBP5
  production
• Aminogylcosides
• Low level to streptocmycin and gentimicin
• Synergism causes cell wall agent to become
  bactericidal
• High level to tobramycin

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Enterococcus-Intrinsic

• Clindamycin-gene encoding efflux pump
• TMP-SXZ-
• In vitro appears susceptible but in vitro is
  resistant
• Can utilize preformed folic acid
• Vancomycin at low levels in some strains

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Enterococcus

• Genetic transfer to acquire new resistance
• One mechanism, involving pheromone-responsive
  plasmids, causes plasmid transfer between E.
  faecalis isolates at a very high frequency .
• Another mechanism involves plasmids that can
  transfer among a broad range of species and
  genera, although usually at a moderately low
  frequency .
• A third mechanism (conjugative transposition)
  involves transfer of specialized transposons at
  low frequency but to a very broad range of
  different kinds of bacteria . Conjugative
  transposons are relatively nonselective in their
  host range and are one of the few types of
  elements known to have crossed the
  gram-positive/gram-negative barrier in naturally
  occurring clinical isolates and to then cause
  resistance in these various hosts

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Enterococcus

• Acquired
• High level resistance to amnioglycosides
• Loose synergy ability as well
• High level vancomycin resistance
• Van gene clusters on transposons or plasmids
• Very old, probably initially resulted from
  pressor from natural glyocpeptides
• Van A is the most common and confers highest
  level of resistance
• Variable level to linezolid
• Depends on the number of mutations in the 23S
  rRNA

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